Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a “bump–hole” chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme–substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme–substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems

Evolving Clustered Random Networks

We propose a Markov chain simulation method to generate simple connected random graphs with a specified degree sequence and level of clustering. The networks generated by our algorithm are random in all other respects and can thus serve as generic models for studying the impacts of degree distributions and clustering on dynamical processes as well as null models for detecting other structural properties in empirical networks

Rheumatoid Factor as a Potentiator of Anti–Citrullinated Protein Antibody–Mediated Inflammation in Rheumatoid Arthritis

Objective. The co-occurrence of rheumatoid factor (RF) and anti–citrullinated protein antibody (ACPA) positivity in rheumatoid arthritis (RA) is well described. However, the mechanisms underlying the potential interaction between these 2 distinct autoantibodies have not been well defined. The aim of this study was to evaluate the epidemiologic and molecular interaction of ACPAs and RF and its association with both disease activity and measures of RA-associated inflammation. Methods. In a cohort of 1,488 US veterans with RA, measures of disease activity and serum levels of cytokines and multiplex ACPAs were compared between the following groups of patients: double-negative (anti–cyclic citrullinated peptide [anti-CCP]-/RF-), anti-CCP+/RF-, anti-CCP-/RF+, or double-positive (anti-CCP+/RF+). Additional studies were performed using an in vitro immune complex (IC) stimulation assay in which macrophages were incubated with ACPA ICs in the presence or absence of monoclonal IgM-RF, and tumor necrosis factor α production measured as a readout of macrophage activation. Results. Compared with the double-negative subgroup (as well as each single-positive subgroup), the double-positive subgroup exhibited higher disease activity as well as higher levels of C-reactive protein and inflammatory cytokines (all P \u3c 0.001). In vitro stimulation of macrophages by ACPA ICs increased cytokine production, and the addition of monoclonal IgM-RF significantly increased macrophage tumor necrosis factor α production (P = 0.003 versus ACPA ICs alone). Conclusion. The combined presence of ACPAs and IgM-RF mediates increased proinflammatory cytokine production in vitro and is associated with increased systemic inflammation and disease activity in RA. Our data suggest that IgM-RF enhances the capacity of ACPA ICs to stimulate macrophage cytokine production, thereby providing a mechanistic link by which RF enhances the pathogenicity of ACPA ICs in RA

Anti-plasmodial polyvalent interactions in Artemisia annua L. aqueous extract – possible synergistic and resistance mechanisms

Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L-1), dihydroartemisinic acid (70.0±0.3 mg L-1), arteannuin B (1.3±0.0 mg L-1), isovitexin (105.0±7.2 mg L-1) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B’s potentiation of artemisinin

Ethnicity-specific factors influencing childhood immunisation decisions among Black and Asian Minority Ethnic groups in the UK: a systematic review of qualitative research

Background: Uptake of some childhood immunisations in the UK is lower among those from some Black and Asian Minority Ethnic (BAME) backgrounds. This systematic review of qualitative research sought to understand the factors that are associated with ethnicity that influence the immunisation decisions of parents from BAME backgrounds living in the UK. Methods: Databases were searched on 2 December 2014 for studies published at any time using the terms ‘UK’ and ‘vaccination’ and ‘qualitative methods’ (and variations of these). Included articles comprised participants who were parents from BAME backgrounds. Thematic synthesis methods were used to develop descriptive and higher order themes. Themes specific to ethnicity and associated factors are reported. Results: Eight papers were included in the review. Most participants were from Black (n=62) or Asian (n=38) backgrounds. Two ethnicity-related factors affected immunisation decisions. First, factors that are related to ethnicity itself (namely religion, upbringing and migration, and language) affected parents' perceived importance of immunisations, whether immunisations were permitted or culturally acceptable and their understanding of immunisation/the immunisation schedule. Second, perceived biological differences affected decision-making and demand for information. Conclusions: Factors related to ethnicity must be considered when seeking to understand immunisation decisions among parents from BAME backgrounds. Where appropriate and feasible, vaccination information should be targeted to address beliefs about ethnic differences held by some individuals from some BAME backgrounds

The clustering of galaxies in the SDSS-III Baryon Oscillation Spectroscopic Survey: Baryon Acoustic Oscillations in the Data Release 9 Spectroscopic Galaxy Sample

We present measurements of galaxy clustering from the Baryon Oscillation Spectroscopic Survey (BOSS), which is part of the Sloan Digital Sky Survey III (SDSS-III). These use the Data Release 9 (DR9) CMASS sample, which contains 264,283 massive galaxies covering 3275 square degrees with an effective redshift z=0.57 and redshift range 0.43 < z < 0.7. Assuming a concordance Lambda-CDM cosmological model, this sample covers an effective volume of 2.2 Gpc^3, and represents the largest sample of the Universe ever surveyed at this density, n = 3 x 10^-4 h^-3 Mpc^3. We measure the angle-averaged galaxy correlation function and power spectrum, including density-field reconstruction of the baryon acoustic oscillation (BAO) feature. The acoustic features are detected at a significance of 5\sigma in both the correlation function and power spectrum. Combining with the SDSS-II Luminous Red Galaxy Sample, the detection significance increases to 6.7\sigma. Fitting for the position of the acoustic features measures the distance to z=0.57 relative to the sound horizon DV /rs = 13.67 +/- 0.22 at z=0.57. Assuming a fiducial sound horizon of 153.19 Mpc, which matches cosmic microwave background constraints, this corresponds to a distance DV(z=0.57) = 2094 +/- 34 Mpc. At 1.7 per cent, this is the most precise distance constraint ever obtained from a galaxy survey. We place this result alongside previous BAO measurements in a cosmological distance ladder and find excellent agreement with the current supernova measurements. We use these distance measurements to constrain various cosmological models, finding continuing support for a flat Universe with a cosmological constant.Comment: 33 page

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a “bump–hole” chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme–substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme–substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems

Exome sequencing identifies novel AD-associated genes

The genetic component of Alzheimer’s disease (AD) has been mainly assessed using Genome Wide Association Studies (GWAS), which do not capture the risk contributed by rare variants. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals —16,036 AD cases and 16,522 controls— in a two-stage analysis. Next to known genes TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Next to these genes, the rare variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential driver genes in AD-GWAS loci. Rare damaging variants in these genes, and in particular loss-of-function variants, have a large effect on AD-risk, and they are enriched in early onset AD cases. The newly identified AD-associated genes provide additional evidence for a major role for APP-processing, Aβ-aggregation, lipid metabolism and microglial function in AD

Effects of Ploidy and Recombination on Evolution of Robustness in a Model of the Segment Polarity Network

Many genetic networks are astonishingly robust to quantitative variation, allowing these networks to continue functioning in the face of mutation and environmental perturbation. However, the evolution of such robustness remains poorly understood for real genetic networks. Here we explore whether and how ploidy and recombination affect the evolution of robustness in a detailed computational model of the segment polarity network. We introduce a novel computational method that predicts the quantitative values of biochemical parameters from bit sequences representing genotype, allowing our model to bridge genotype to phenotype. Using this, we simulate 2,000 generations of evolution in a population of individuals under stabilizing and truncation selection, selecting for individuals that could sharpen the initial pattern of engrailed and wingless expression. Robustness was measured by simulating a mutation in the network and measuring the effect on the engrailed and wingless patterns; higher robustness corresponded to insensitivity of this pattern to perturbation. We compared robustness in diploid and haploid populations, with either asexual or sexual reproduction. In all cases, robustness increased, and the greatest increase was in diploid sexual populations; diploidy and sex synergized to evolve greater robustness than either acting alone. Diploidy conferred increased robustness by allowing most deleterious mutations to be rescued by a working allele. Sex (recombination) conferred a robustness advantage through “survival of the compatible”: those alleles that can work with a wide variety of genetically diverse partners persist, and this selects for robust alleles